EP3574120A1 - Obtention de souches de levure performantes pour la metabolisation de l'arabinose - Google Patents
Obtention de souches de levure performantes pour la metabolisation de l'arabinoseInfo
- Publication number
- EP3574120A1 EP3574120A1 EP18712080.3A EP18712080A EP3574120A1 EP 3574120 A1 EP3574120 A1 EP 3574120A1 EP 18712080 A EP18712080 A EP 18712080A EP 3574120 A1 EP3574120 A1 EP 3574120A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- arabinose
- strain
- xylose
- medium
- advantageously
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Classifications
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/85—Saccharomyces
- C12R2001/865—Saccharomyces cerevisiae
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Definitions
- the present invention relates to yeast strains capable of metabolizing arabinose, advantageously in combination with their ability to ferment xylose and glucose, including in the presence of inhibitors of these last two fermentations, such as acetic acid. in non-dissociated form.
- the present invention provides a method for selecting strains capable of metabolizing arabinose and improved strains, performing in their ability to metabolize arabinose but also xylose, both types of pentoses being found in lignocellulosic hydrolysates .
- Lignocellulosic or plant biomass essentially derived from agricultural and agro-industrial activity, is a complex substrate made up of three main fractions: cellulose, hemicelluloses and lignin. It is a recyclable waste, useful for the production of ethanol whose demand, for example for its use as a biofuel, continues to grow.
- the process for producing ethanol from lignocellulosic biomass consists in recovering by hydrolysis the maximum of sugars present in the cellulosic and hemicellulosic fractions and then transforming them into ethanol by fermentation.
- yeasts strains capable of fermenting glucose have been modified to also be able to metabolize pentoses.
- the document WO 2010/000464 reports the production of yeast strains capable of fermenting pentoses by means of a bacterial gene encoding a xylose isomerase (XI) which converts xylose into yeast-metabolizable xylulose.
- XI xylose isomerase
- a pathway comprising xylose reductase (XR or XYL1) generating xylitol and xylitol dehydrogenase (XDH or XYL2) also leads to xylulose.
- WO 2012/072793 describes improved yeast strains, combining exogenous genes encoding a xylose isomerase and a xylitol dehydrogenase to remove xylitol which is found to be a xylose isomerase inhibitor.
- Such strains in particular the strain deposited at the CNCM 5.10.2011 under the number I-4538, have improved yields and therefore a proven industrial utility for the production of ethanol.
- acetic acid is also an inhibitor of xylose fermentation. This inhibition is characterized by a reduction in the kinetics of consumption of xylose (Bellisimi et al., FEMS Yeast Res., 2009, 9: 358-364), whereas on glucose, this inhibition results in a delay in the initiation of the fermentation, the kinetics remaining thereafter unchanged.
- yeast strains primarily ferment glucose due to catabolic repression.
- the documents WO 2013/178915 and WO 2013/178918 describe processes for obtaining yeast strains capable of metabolizing pentoses, in particular xylose, and resistant to fermentation inhibitors, in particular acetic acid. Strains further improved, particularly in their ability to ferment glucose and xylose in the presence of acetic acid, are described in WO 2015/121595 and FR 3,035,405.
- the residual arabinose can be a substrate of choice for the development of contaminating microorganisms, possibly capable of converting it into organic acids which, as already said, are inhibitors of glucose and xylose fermentation (Schell et al., 2007, Bioresour, Technol 98, 2942-2948).
- microorganisms have been described as being able to metabolize arabinose in both fungal microorganisms, especially hemi-ascomycetes such as Scheffersomyces stipitis, basidiomycetes and filamentous fungi, as well as in bacteria such as Erwinia chrysanthemi, Thermoanaerobacterium saccharolyticum , Escherichia coli, Zymomonas mobilis, Bacillus subtilis, Bacillus licheniformis and Lactobacillus plantarum.
- hemi-ascomycetes such as Scheffersomyces stipitis, basidiomycetes and filamentous fungi
- bacteria such as Erwinia chrysanthemi, Thermoanaerobacterium saccharolyticum , Escherichia coli, Zymomonas mobilis, Bacillus subtilis, Bacillus licheniformis and Lactobacillus plantarum.
- WO 03/095627 has reported the possibility of obtaining strains of S. cerevisiae capable of growing on arabinose by transforming a laboratory strain by multicopy plasmids specifically carrying the araA genes (coding an L-arabinose isomerase) of B. subtilis, araB (encoding a favorably mutated L-ribulokinase) and araD (encoding an L-ribulose-5-P-4 epimerase) of E. coli. coli.
- This document openly raises the question of the possibility of obtaining a strain capable of co-fermenting xylose.
- WO 2008/122354 discloses strains of S. cerevisiae transformed with self-replicative vectors carrying the araA genes of B. licheniformis, araB (advantageously mutated) and AraD of E. coli, advantageously codon-optimized, able to grow aerobically or anaerobically in media containing as sole carbon source arabinose.
- Transformants are selected by culturing in a medium containing glucose as a carbon source.
- the document WO 2012/143513 reports the chromosomal integration of at least the araA, araB and araD and XylA genes into a yeast strain of the Saccharomyces type which, after culturing according to the "Sequential Batch Repeat" method as described in WO 2009/112472 in media containing 20g / L arabinose and 20g / L xylose, gives it the ability to ferment glucose, xylose and arabinose. Mutations in different genes (SSY1, YJR154w, CEP 3, GAL80, PMR1) have been highlighted.
- the present invention is part of the search for new yeast strains capable of metabolizing at least arabinose, based on the contribution of the inventors in connection with various aspects:
- yeast strains having an improved ability to ferment both arabinose, xylose and glucose, including in the presence of inhibitors of the fermentation of these last two sugars such as acetic acid.
- the present invention relates to a process for obtaining and / or selecting a yeast strain capable of metabolizing arabinose, comprising the following steps:
- this method comprises:
- this method furthermore comprises the selection of transformed strains which have metabolized arabinose on the basis of their aldose reductase activity of less than or equal to 0.002 U / g of protein, or even less than or equal to 0.0005 U / g. of protein.
- yeast strain means a population of yeasts rigorously identical from the genetic point of view. This includes both so-called laboratory strains and so-called industrial strains.
- the yeast strain used in the context of this process is chosen from among Saccharomyces, Schizosaccharomyces, Pichia, Yarrowia, Paffia, Kluyveromyces, Candida, Talaromyces, Brettanomyces, Pachysolen, Hansenula, Kloeckera, Schwanniomyces and Debaryomyces, advantageously a strain of Saccharomyces cerevisiae.
- yeasts are known not to be able to naturally metabolize arabinose or at a very low level, not exploitable industrially. They are also advantageously chosen for their ability to carry out anaerobic fermentation, even more advantageously anaerobic alcoholic fermentation. According to a particular embodiment, the process, which is the subject of the present application, is carried out on a strain capable of fermenting xylose, advantageously capable of fermenting xylose in the presence of an organic acid in undissociated form, such as 'acetic acid.
- the ability to metabolize xylose may result from the introduction of a gene encoding xylose isomerase, for example from Clostridium phytofermentans, and / or xylulokinase.
- a yeast strain capable of metabolizing arabinose is a strain capable of consuming or using arabinose, advantageously L-arabinose, present in its culture medium.
- a yeast strain can use arabinose for the production of its biomass and / or the generation of fermentation products.
- the fermentation of arabinose is understood as the metabolisation of arabinose taking place in hypoxia and / or in anaerobiosis, that is to say in conditions of low availability (typically less than 20%) or total absence of oxygen.
- the term "metabolize” can therefore refer both to the ability of the yeast strain to use arabinose to ensure its growth and its ability to ferment arabinose in various fermentation products such as derivatives. hydroxyls, including ethanol or isobutanol, and / or carboxylated, including organic acids.
- L-arabinose is converted to L-ribulose by the action of an arabinose isomerase (araA, EC: 5.3.1.4).
- L-ribulose can itself be converted to L-ribulose-5-phosphate by ribulokinase (araB, EC: 2.7.1.16).
- L-ribulose-5-phosphate can then be converted to D-xylulose-5-phosphate by ribulose phosphate epimerase (araD, EC: 5.1.3.4).
- the D-xylulose-5-phosphate is then supported by the non-oxidative part of the pentose phosphate pathway and can lead to the production of ethanol.
- the gene (s) allowing the metabolization of arabinose are introduced into the yeast strain, which is then called transformant.
- the terms "gene” or “sequence” refer to a nucleic acid sequence comprising a coding sequence (coding in particular an enzyme of the pathway of interest), optionally flanked by regulatory sequences, in particular a promoter or terminator.
- a coding sequence can be optimized, that is to say modified to integrate the preferential codons of the host, in this case the yeast, in which this sequence is to be expressed.
- the genes encoding the arabinose metabolizing pathway are so-called exogenous or heterogeneous genes, which may be of a synthetic nature or from other organisms (or sources).
- they come from microorganisms able to metabolize arabinose, advantageously hemi-ascomycetes such as Scheffersomyces stipitis, basidiomycetes, filamentous fungi or bacteria such as Erwinia chrysanthemi, Thermoanaerobacterium saccharolyticum, Escherichia coli, Zymomonas mobilis, Bacillus subtilis, Bacillus licheniformis or Lactobacillus plantarum.
- these genes come from the bacterium Bacillus licheniformis and / or Escherichia coli. According to another preferred embodiment, these genes correspond to the araA gene of B. licheniformis, to the araB gene of E. coli and the araD gene of E. coli, as described by Widemann and Boles (2008, Applied Environmental Microbiology 74, 2043-2050, WO 2008/122354).
- Techniques for introducing DNA into a host are well known to those skilled in the art and include permeabilization membranes by applying an electric field (electroporation), thermally (application of a thermal shock) or chemically, for example using lithium acetate.
- the introduced genes can be integrated into the genome of the host, in particular by homologous recombination or chromosomal integration, advantageously using integrative cassettes, or expressed extrachromosomally using plasmids or vectors.
- plasmids advantageously self-replicating, are well known to those skilled in the art, which differ in particular by their origin of replication, their promoter (inducible or constitutive), their marker (for example a resistance to an antibiotic or the ability to grow in a selective medium) and the number of copies per cell.
- the genes encoding the metabolization pathway of arabinose are integrated at the chromosomal level, advantageously at the HO locus of the yeast strain, still more advantageously at all the HO loci of the yeast strain. .
- the cassette carrying this gene (s) and serving for their incorporation or integration into the yeast strain is devoid of a marker, especially antibiotic resistance.
- the cassette carrying this gene (s) does not carry a gene encoding an arabinose transporter, in particular the araT gene.
- the correct introduction of the gene or genes can easily be verified by the techniques known to those skilled in the art, for example with the aid of the marker possibly carried by the expression cassette, or, preferably, by carrying out a PCR at the same time. using primers targeting the introduced gene (s).
- this same PCR technique can be used to verify the integration of the gene or genes at the target locus, in particular by using primers targeting this locus.
- a strain of interest having incorporated and effectively expressing the arabinose metabolizing pathway is selected for its ability to metabolize the arabinose present, as sole carbon source, in the medium. of culture.
- the selection of the transformants is done directly on a medium containing arabinose as sole source of carbon.
- a yeast strain to metabolize arabinose is tested by culturing said strain on a medium containing, as sole source of carbon, said arabinose. This precludes any prior induction, in particular a prior culture in the presence of galactose or the addition of galactose in this medium, or a preselection of the transformants on a medium containing another carbon source such as glucose.
- a culture or growth medium designates a medium comprising the ingredients necessary for the multiplication of yeasts in the presence. It is advantageously a complete medium, suitable for the growth of yeast and may contain conventional ingredients such as salts, buffering agents, yeast extract or any other source of nitrogen metabolizable by yeast, vitamins... .
- synthetic medium means a medium whose chemical composition is known.
- the culture conditions used are culture conditions favorable to the growth of yeasts, in particular:
- a culture medium at acidic pH advantageously between 4 and 6, or even 4.5 and 5.5, still more advantageously equal to 5 or 5.4;
- this growth is carried out in solid medium, which makes it possible to isolate the strains actually capable of metabolizing arabinose.
- the selection of the strains of interest is carried out by means of Petri dishes.
- the solid media contain agar, advantageously up to 15 to 20 g / l.
- a yeast strain capable of metabolizing arabinose is selected by aerobic growth of this strain in a growth medium containing, as sole source of carbon, arabinose.
- a suitable medium for this aerobic growth is the YNB Difco ® synthetic medium, the exact composition of which is given in the exemplary embodiments, comprising arabinose as sole source of carbon, advantageously up to 10 g / l, such as medium called YNB-Ara below.
- a yeast strain capable of metabolizing arabinose can be selected by growth in anaerobiosis or hypoxia in a growth medium containing, as sole source of carbon, arabinose.
- a suitable medium for this growth in anaerobiosis or hypoxia is, for example, the synthetic medium YF, the exact composition of which is given in the exemplary embodiments, comprising arabinose as sole source of carbon, advantageously up to 70 g / l. , such as the medium called YF-ara hereinafter.
- this medium is adapted to allow growth in a solid medium, for example by adding agar, thus making it possible to directly isolate the strains of interest.
- the yeast strains into which the arabinose metabolizing pathway has been introduced are isolated by culture in solid medium, advantageously on a selective medium such as the YNB-Ara medium described above, then selected in a liquid medium, anaerobically or hypoxia, in a suitable medium such as the YF-ara medium described above.
- a selective medium such as the YNB-Ara medium described above
- a suitable medium such as the YF-ara medium described above.
- These liquid cultures can be carried out in Deep Weel-type microplates or in vials, with gentle stirring, for example equal to 100 rpm, or without stirring and under reduced oxygen supply conditions (limited 0 2 or anaerobic ). Under these conditions, the capacity of yeast strains to metabolize arabinose can be evaluated by:
- OD optical density
- the yeast strains thus obtained can be further tested, in parallel or subsequently, under the following conditions:
- a growth medium containing glucose as sole source of carbon for example on a YF medium as described above but containing 150 g / l of glucose, and under the culture conditions mentioned above.
- This step makes it possible to check the validity of the selected strains, in particular their ability to ferment glucose. Note that one or more passages on glucose medium can be performed before "ironing" the selected strains on a medium containing arabinose as sole source of carbon (as described above) and to verify that the phenotype [ara + ] is stable and conserved;
- a growth medium containing xylose as sole source of carbon for example on a YF medium as described above but containing 70 g / l of xylose (YF-xylose medium in the embodiments), and under the conditions of culture mentioned above.
- This step makes it possible to verify that the selected strains have retained their ability to ferment xylose and is of interest when the yeast strain on which the process of the invention is carried out has an ability to ferment xylose.
- an important criterion for the selection of strains of interest for the metabolization of arabinose was their level of polyol production.
- polyols such as xylitol have an inhibitory effect on xylose isomerase (xylA) activity (Kovalevsky et al., 2012, Acta Crystallogr D Biol Crystallogr 68, 1201-1206).
- xylA xylose isomerase
- the polyols are also capable of inhibiting arabinose isomerase activity (araA; FIG. 1), and thus of decreasing the performance, in terms of the metabolism of arabinose. of a strain selected using the method according to the invention.
- Arabitol is likely to be generated from arabinose under the action of aldose reductase (s), in the same way that xylitol is generated from xylose.
- aldose reductase s
- the GRE3 gene codes for the main aldose reductase in S. cerevisiae, but that even when this is deleted or inactivated, an aldose reductase activity may persist.
- the presence in the yeast strains of a xylitol dehydrogenase activity can potentially eliminate xylitol.
- a selected strain of particular interest has:
- a high polyol dehydrogenase activity advantageously greater than or equal to 0.001 U / g of protein, still more advantageously greater than or equal to 0.002 U / g of protein;
- a strain of interest could be selected because of its low or even zero aldose reductase activity.
- this activity is less than 0.005 U / g of protein, or even 0.004, 0.003 or even 0.002 U / g of protein. More preferably, it is less than or equal to 0.0015 U / g of protein, or even less than or equal to 0.001 U / g of protein, still more advantageously less than or equal to 0.0005 U / g of protein.
- the yeast strain used in the context of the method according to the invention has one or more GRE3 gene (s) deleted or inactivated (s).
- a yeast strain used in the context of the present invention has at least one supernumerary copy of the HAA1 gene encoding the transcriptional regulator Haalp.
- yeast strains in particular S. cerevisiae, a capacity to resist acetic acid and thus makes it possible to improve their growth in a medium containing inhibitors of type fermentation. organic acid, such as acetic acid.
- organic acid such as acetic acid.
- the applicant has demonstrated that the overexpression of this gene conferred resistance to other inhibitors of the phenolic compounds type, in particular vanillin.
- the expression of the supernumerary gene encoding Haalp is placed under the control of a heterologous promoter, for example the pPGK1 promoter.
- This supernumerary copy of the HAA1 gene can encode the native protein or a mutated form thereof, for example the constitutively active version HAAIS SF described by Swinnen et al. (2017, Microbial Cell Factories 16: 7).
- the additional copy of the HAA1 gene is integrated at the chromosomal level, still more advantageously by insertion at the level of the GRE3 gene.
- the overexpression of the HAA1 gene conferring greater resistance of the yeast strains to acetic acid but also to vanillin.
- the second step of the process according to the invention can implement the evaluation of one or the other of the above two criteria, advantageously 2.
- the ability to metabolize arabinose present in the culture medium is first evaluated then the selected strains are tested for their activity aldose reductase.
- an acidic and stable pH advantageously between 4 and 6, for example equal to 5;
- a temperature between 28 and 37 ° C, or between 30 and 35 ° C, preferably equal to 30 ° C;
- the cultivation may be performed in a vial sealed with a stopper reducing the supply of 0 2 in the middle while allowing the evacuation of the C0 2 produced;
- the present invention relates to a yeast strain obtainable by the method described, capable of fermenting at least 50%> or even 60%>, 70%> or even 80%> arabinose after 72 hours of fermentation in a medium comprising glucose (advantageously at a level of 1 to 100 g / l, for example 63 g / l), xylose (advantageously at a level of 1 to 100 g / l, example 28 or 52 g / L), arabinose (advantageously at 1 to 100 g / L, for example 6.1 or 28 g / L) and acetic acid (advantageously at a level of 1 to 10 g / l).
- g / L for example 4 g / L
- such a yeast strain is capable of fermenting at the same time at least 90%> or even 95% of the glucose and xylose also present.
- Such a strain advantageously also has at least one of the following characteristics, or all these characteristics:
- At least one copy of an araA gene preferably from B. licheniformis, advantageously integrated at the chromosomal level, still more advantageously at the level of at least one HO locus;
- At least one copy of an araB gene preferably from E. coli. coli, advantageously integrated at the chromosomal level, still more advantageously at the level of at least one HO locus;
- At least one copy of a araD gene preferably from E. coli. coli, advantageously integrated at the chromosomal level, still more advantageously at the level of at least one HO locus;
- At least one copy of an exogenous gene encoding a xylose isomerase preferably Clostridium phytofermentans
- said strain comprises at least two supernumerary copies of the GAL2 gene. This can be placed under the control of a strong and constitutive promoter of pADH1 type; the suppression of the aldose reductase activity encoded by GRE3, advantageously by insertion of the HAA1 gene at the GRE 3 locus;
- XKS1 xylulokinase
- RPE1 pentose phosphate pathway
- such a strain is not mutated at the level of the following genes: SSY1, YJR154w, CEP 3, GAL80 and / or PMR1.
- the strain does not carry the following mutations: G1363T in the SSY1 gene, A512T in the YJR154w gene, A1186G in the CEP3 gene, A436C in the GAL80 gene, Al 13G in the PMR1 gene.
- a strain of particular interest, obtained by the implementation of the process claimed, is the strain deposited with the CNCM (National Collection of Cultures of Microorganisms, Pasteur Institute, 25 rue du Dondel Roux, 75724 Paris Cedex 15) dated May 19, 2016, under the number 1-5085.
- the invention relates to a method for obtaining and / or selecting yeast strains having an improved ability to ferment arabinose, xylose and glucose, including in the presence of fermentation inhibitors of the type organic acid, especially acetic acid, resulting in particular in an increase in ethanol production and a greater consumption of arabinose present in the medium.
- the invention also relates to a process for obtaining and / or selecting a yeast strain having an improved capacity to ferment glucose, xylose and arabinose, advantageously in the presence of an organic acid in non-organic form.
- dissociated such as acetic acid
- a yeast strain having such a capacity is cultivated successively under the following conditions:
- Such a process therefore consists in carrying out a directed evolution of the yeast strains used.
- the selection pressure exerted by the successive cultures in the growth media defined below makes it possible to select a strain having acquired the phenotypic traits necessary to increase its capacity to ferment arabinose. while maintaining its ability to ferment xylose and glucose, including in the presence of organic acid in undissociated form, particularly acetic acid.
- the method according to the invention makes it possible, from an isolated strain or a mixture of strains, to select a strain having a selective advantage in terms of growth on a medium containing these three sugars and said organic acid in the form of not dissociated.
- This method can be implemented on an isolated strain, in particular on the strain deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15) dated May 19, 2016, under the number 1-5085.
- CNCM National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Dondel Roux, 75724 Paris Cedex 15
- the yeast strain or a possible mixture of strains is cultivated successively in at least three growth media.
- growth medium or “culture medium” are used interchangeably to designate a medium comprising the ingredients necessary for the multiplication of the yeasts present.
- the first growth medium advantageously liquid, is characterized in that it comprises, as sole carbon sources, pentoses arabinose and xylose.
- this first medium comprises a concentration of pentoses making it possible to cover together 100%, advantageously each 50%, of the carbon requirements of the yeast strains.
- arabinose and xylose are present at equivalent concentrations, for example at 5 g / L for arabinose and 4 g / L for xylose.
- it is a synthetic medium whose exact composition is controlled, advantageously a medium whose chemical composition is entirely determined.
- a medium is for example the medium called "GO xylose arabinose" whose precise composition is given in the embodiments.
- it is not the nitrogen source is limiting for biomass (10 g / L of (NH BP0) but the amounts of carbon sources, namely xylose (4 g / L) and arabinose (5 g / L)
- a medium promotes the growth and multiplication of yeast strains that use both sugars.
- the next step is to anticipate a possible risk of loss of tolerance vis-à-vis the inhibitors, particularly acetic acid in undissociated form, selected strains.
- the order of passage on these two culture media can optionally be reversed (xylose medium and glucose).
- the glucose or xylose concentration of the growth medium is that commonly used when it is used as the only source of carbon, namely between 5 and 200 g / l in the case of a medium.
- said medium further comprises the organic acid capable of inhibiting the fermentation of these two sugars.
- organic acid capable of inhibiting the fermentation of these two sugars.
- acetic acid or formic acid more advantageously acetic acid.
- acetic acid formic acid
- acetic acid more advantageously acetic acid.
- non-ionized or non-dissociated form of such acids has an inhibition capacity.
- non-ionized or non-dissociated form of a carboxylic acid its protonated form.
- the form of such organic acids depends on the pH of the medium in which they are incorporated. At a pH above the pKa of the acid, it is principally present in dissociated form or COO ions ".
- the major form is the undissociated form or unionized (COOH).
- COOH unionized
- the concentration of organic acid in undissociated form, advantageously in acetic acid, of the growth medium is between 0.5 and 5 g / L in a liquid medium (equivalent to 0.5 and 5 g / kg in medium). solid), advantageously between 1.3 and 2.6 g / l.
- this last range corresponds to a concentration of acetic acid added to a growth medium at pH 5 of between 3 and 6 g / l, for example equal to 4 or 5 g / l.
- this growth medium is advantageously a complete synthetic medium, suitable for the growth of yeasts in anaerobiosis or hypoxia, such as, for example, YF medium, the exact composition of which is given in the exemplary embodiments.
- suitable media for the implementation of this second step of the process according to the invention are for example the media called YF-glucose acid and YF-xylose acid, whose composition is detailed in the embodiments.
- the culture of the yeast strain or the mixture of strains in these first and second stages of the process according to the invention is advantageously carried out under standard conditions favorable to the growth in anaerobiosis or hypoxia of yeasts, in particular of the Saccharomyces type, and their fermentation activity, namely:
- an acidic pH advantageously between 4 and 6, even 4.5 and 5.5, still more advantageously equal to 5 or 5.4;
- a culture under slight agitation for example equal to 100 rpm; under reduced conditions of oxygen supply (limited 0 2 ).
- the culture can be performed in a vial closed with a plug reducing the supply of 0 2 in the medium while allowing the evacuation of C0 2 produced.
- the culture is stopped when the source of carbon dioxide has been totally consumed.
- the culture is conducted for at least 24 hours, or even several days, advantageously up to 7 days.
- the method according to the invention further comprises the passage of yeasts on a fourth growth medium, preferably liquid, for selecting the cells capable of breathing, namely having functional mitochondria.
- a fourth growth medium preferably liquid
- this step which can be implemented at each cycle or at least once in the process, eliminates the appearance of "small", whose respiratory deficiency phenotype can be disadvantageous in the context of production processes of industrial yeasts.
- this fourth medium is a poor or minimum medium, containing as a sole source of carbon a carbon source that can only be used by cells that have retained functional mitochondria.
- a source of strict respiratory carbon ie a carbon source systematically involving a mitochondrial oxidation and not producing ethanol. It may be advantageously glycerol or possibly ethanol.
- such a medium is devoid of fermentable sugar.
- the concentration of glycerol of the growth medium is that commonly used when it is used as the only source of carbon, namely between 5 and 50 g / l, advantageously between 10 and 50 g / l, by example equal to 10 g / L so as to obtain a biomass sufficient to possibly inoculate the first culture medium of the next cycle.
- a minimum medium contains, besides a source of carbon, a source of nitrogen, a source of potassium, a source of phosphorus, a source of sulfur, a source of magnesium, a source of calcium, a source of iron, a source of trace elements and water.
- An environment that can be used to develop this culture medium may include:
- a base such as "yeast nitrogen base” DIFCO ®, preferably up to 3.4 g / L;
- ammonium sulphate advantageously up to 5 g / l.
- the culture of the yeast strain or the mixture of strains is advantageously carried out under standard conditions favorable to the growth of yeasts under aerobic conditions, namely:
- an acidic pH advantageously between 4 and 6, or even 4.5 and 5.5, for example equal to 5;
- the culture can be carried out in a baffle flask closed by a porous plug which allows the supply of 0 2 in the medium.
- the culture is stopped when the carbon source, advantageously glycerol, has been totally consumed.
- the culture is conducted for several hours, advantageously 48 hours.
- these different cultures are produced according to the so-called “Single Batch Repeat” method, that is to say without renewal of the culture media in the presence.
- such a cycle is repeated at least twice ("Multiple Batch Repeat"), for example 2 times.
- This process has allowed the selection of yeast strains which, during the fermentation on a medium close to the natural media including in particular arabinose, glucose, xylose and acetic acid, have improved ethanol production ( a higher alcoholic strength) and a better consumption of arabinose present in the medium.
- the present invention therefore relates to a yeast strain obtainable by means of the method described.
- yeast strains which, at the end of this selection process, have a capacity of consumption of arabinose present in the medium increased by at least 10%, or even 20%, 30% , 40%, 50%, 60%, 70%, even 80%.
- the present invention relates to a yeast strain obtainable by the method described, capable of fermenting at least 90%> or even 95%>, or even 97.5%> of arabinose after 160 hours of fermentation in a medium comprising glucose (advantageously from 1 to 100 g / l, for example 63 g / l), xylose (advantageously from 1 to 100 g / l, for example 28 g / L), arabinose (advantageously at a level of 1 to 100 g / l, for example 28 g / l) and acetic acid (advantageously at a level of 1 to 10 g / l, for example 4 g / L).
- a yeast strain is capable of fermenting at the same time at least 90%> or even 95%> or even 100%> glucose and xylose also present.
- such a strain has at least one of the following characteristics, or all these characteristics:
- At least one copy of a araA gene preferably from B. licheniformis, advantageously integrated at the chromosomal level, still more advantageously at all the HO loci; at least one copy of an araB gene, preferably from E. coli. coli, advantageously integrated at the chromosomal level, still more advantageously at all the HO loci;
- At least one copy of a araD gene preferably from E. coli. coli, advantageously integrated at the chromosomal level, still more advantageously at all the HO loci;
- At least one copy of an exogenous gene encoding a xylose isomerase preferably Clostridium phytofermentans
- said strain comprises at least two supernumerary copies of the GAL2 gene. This can be placed under the control of a strong and constitutive promoter of pADH1 type;
- XKS1 xylulokinase
- RPE1 pentose phosphate pathway
- such a strain has at least one of the following characteristics, advantageously the 2:
- a high polyol dehydrogenase activity advantageously greater than or equal to 0.001 U / g of protein, still more advantageously greater than or equal to 0.002 U / g of protein;
- such a strain is not mutated at the level of the following genes: SSYI, YJR154w, CEP 3, GAL80 and / or PMR1.
- the strain does not carry the following mutations: G1363T in the SSYI gene, A512T in the YJR154w gene, A1186G in the CEP3 gene, A436C in the GAL80 gene, Al 13G in the PMR1 gene.
- a strain of particular interest is the strain deposited with the CNCM (National Collection of Cultures of Microorganisms, Pasteur Institute, 25 rue du Dondel Roux, 75724 Paris Cedex 15) dated May 19, 2016, under the number 1-5086.
- such a strain has at least one supernumerary copy of the HAA1 gene, advantageously inserted in the GRE 3 gene.
- the present invention also relates to a yeast obtained by culturing strains as defined above.
- yeast a commercial product obtained through the implementation of a method of producing a yeast strain.
- yeasts having different characteristics can be obtained from the same strain, these differences being related to the production method used.
- the invention relates to the use of strains or yeasts as defined above for the fermentation of a material, advantageously containing arabinose and / or xylose and / or glucose, and / or or for the production of ethanol.
- a material advantageously containing arabinose and / or xylose and / or glucose, and / or or for the production of ethanol.
- the material is a lignocellulosic material.
- Such a material typically contains:
- pentoses in particular D-xylose and L-arabinose
- hexoses in particular D-mannose, D-galactose, L-rhamnose and D-glucose;
- the invention relates to a process for producing fermentation products or ethanol comprising the following steps:
- the material or medium is hemicellulose "corn fbers".
- Figure 1 shows the metabolic pathways of conversion of L-arabinose and D-xylose to D-xylulose-5-phosphate.
- FIG. 2 represents the plasmid pLI285-055 allowing the integration of the ciraAIaraBlaraD genes at the HO locus of Saccharomyces cerevisiae.
- Figure 3 shows the PCR products obtained using the oligonucleotides of Table 1 below.
- Lane 1 corresponds to the size marker (1 kb DNA ladder)
- lanes 2 to 9 correspond to the PCR products obtained using as template the genomic DNA of the 8 transformants tested
- lane 10 to that obtained with the untransformed strain .
- Lane 11 is the negative control of the technique (water)
- lane 12 is the positive control with as template plasmid pLI285-055.
- Figure 4 illustrates the growth of colonies on YPG + blasticidin 50 mg / L medium (A) and on YNB-Ara medium containing 10 g / L arabinose (B).
- Figure 5 illustrates the growth of the selected clones on a medium containing arabinose (A) or xylose (B) as the only carbon source.
- the values represent the average values from 3 biologically independent experiments.
- Figure 7 shows the aldose reductase activity measured in the different genetic backgrounds tested (1-4953, clone 13, clone 126). Values represent mean values from 2 biologically independent experiments.
- Figure 8 shows the loss of mass observed after 48 hours of fermentation in clone 126 and its transformants (transformed with pADH1-GRE3).
- FIG. 9 represents (A) the evolution of the ethanol concentration (g / kg) as a function of the fermentation time on a YFCF medium of different clones selected after a directed evolution carried out from the strain EG31; (B) the concentration of arabinose (g / kg) after 160 hours of fermentation on YFCF medium of these same clones and strain EG31.
- This strain is otherwise known to be able to metabolize xylose due to the presence of at least one copy of an exogenous gene encoding xylose isomerase of Clostridium phytofermentans.
- this strain overexpresses the genes encoding the enzymes of the non-oxidative part of the pentose phosphate pathway, in particular the TAL1 and TKL1 genes.
- araA-v-r-1 (SEQ ID NO: 2) ATGTCAATCTTGTCCCATGG
- araB-v-r-2 (SEQ ID NO: 4) CCACAAAACGAACATAGCGT
- araD-vr-1 (SEQ ID NO: 6) TAGAAGTAGTCAGCGTGGGT The PCR products obtained were analyzed on an agarose gel at 0.8% after migration of one hour at 50 volts in 0.5X TAE.
- the cassette has been modified to express the ara genes A, B and D while being devoid of coding sequence for an arabinose transporter (ara 7).
- strain 1-4953 deposited at the CNCM on January 29, 2015, was transformed using 1.3 ⁇ g of this cassette carrying araA, B and D.
- the transformation product was spread over 2 types of media:
- a first medium corresponding to YPG (10 g / l of yeast extract, 10 g / l of peptone, 20 g / l of glucose as sole source of carbon) containing Blasticidine at a level of 50 mg / l.
- the goal is to determine the number of cells that have integrated the cassette into their genome;
- YNB-Ara a second medium which contains 10 g / L of arabinose as sole source of carbon.
- This medium must make it possible to determine the number of cells that have acquired the [ara +] phenotype.
- the YNB Difco ® medium ("Yeast Nitrogen Base with amino acids and ammonium sulphate”), referenced under the number 0335-15, contains:
- Figure 4 illustrates the result obtained on these two types of media. After 6 days of growth on both media, colonies were counted. It has been observed that the number of colonies on YNB-Ara medium represents approximately 18% of that observed on YPG + Blasticidine. In other words, only 18% of the colonies that have integrated the araA, B and D genes into their genome seem able to grow using arabinose as the only carbon source.
- the cassette has been further simplified and devoid of any selection marker, including antibiotic resistance.
- YF-ara medium YF medium containing 70 g / L of arabinose
- YF-xylose medium YF medium containing 70 g / L of xylose
- the pH of the medium is maintained at 5.
- the culture is carried out at 32 ° C.
- FIG. 5 illustrates the results obtained with a series of strains, the growth of which was followed in parallel on a medium containing arabinose (A) or xylose (B) as sole source of carbon, the latter medium making it possible to select the strains that have retained their ability to metabolize xylose.
- FIG. 5A shows that 15 clones, among the 91 tested, do not seem able to start multiplying again using the arabinose. This suggests that, unlike selection based on antibiotic resistance, selection on YNB-Ara yields 85% of transformants that have acquired the [ara +] phenotype.
- Another component derived from the results presented in FIG. 5 concerns the coexistence of the two xylose and arabinose metabolic pathways. Thus, it is apparent that some strains acquired the [ara +] phenotype at the expense of the [xyl +] phenotype.
- clones 13 and 126 were compared during the fermentation in YFCF medium at pH 5 comprising as a carbon source glucose, but also arabinose and xylose equi-represented, as well as acetic acid, thus getting closer to natural fermentation media.
- composition of the YFCF medium is as follows:
- the fermentation conditions are as follows:
- Preculture / Incubation 0.25 g / kg eq MS yeasts previously propagated to saturation on YPG medium for 24 hours.
- the production of ethanol is indirectly estimated by measuring the loss of mass of the fermentation flask, this loss of mass being directly correlated with the production of CO2 which is stoichiometric with that of alcohol. It is expressed in g per kg of medium.
- the concentrations of glucose, xylose, arabinose and glycerol in the medium are monitored by HPLC.
- Biomass variations are evaluated by measuring the residual dry matter after 4 hours at 105 ° C.
- clone 13 consumed 12.1 +/- 0.6 g / kg of arabinose, while the clone 126 consumed 15.3 +/- 0.5 g / kg.
- the strains to be tested are grown in rich medium (YPG). Two successive propagation phases of 24 to 30 ° C, with stirring, are necessary to obtain sufficient biomass. Once the biomass harvests have been carried out, the measurement of the dry matter is carried out on 1 ml of each cream after passing in an oven at 105 ° C. for 4 hours.
- the fermentations are carried out in 250 ml flasks containing 100 g of YF-xylose fermentation medium. Each flask is seeded at a rate of 0.5 g / kg dry matter equivalent.
- the fermentations are carried out at 32 ° C., with stirring at 110 rpm and last for 3 days. At this stage, 50 ml of fermentation product are collected and then used for the determination of the different enzymatic activities and the protein concentration of the sample. Preparation of the cell extract
- 1 ml resuspended pellet is then transferred to 1 tube for Fast Prep (previously filled with 250 ⁇ of beads) and then ground at 4 ° C: 4 x 30 seconds (6 m / s), spaced 30 seconds each. Then, the ground material is centrifuged for 3 minutes at 10000 rpm (to drop the beads as well as cell debris) and all of the supernatant is transferred to an ice-cooled Eppendorf tube.
- the enzymatic activity is assayed by monitoring the absorbance at 340 nm.
- the measurements are carried out in a thermostated environment at 32 ° C.
- the GRE3 gene which codes for the principal aldose reductase in S. cerevisiae yeast (Garay-Arroyo and Covarrubias, 1999, Yeast 15, 879-892, Trâff et al., 2001 Appl., Environ., Microbiol. 5668-5674) has been reintroduced.
- a copy of the GRE3 gene placed under the control of the promoter of the ADH1 gene has been integrated at the level of the native GRE 3 locus in the genome of clone 126.
- strain EG31 which has been deposited with the Pasteur Institute (National Collection of Cultures of Microorganisms, 25 rue du Dondel Roux, 75724 Paris Cedex 15) under the number CNCM 1-5085 dated May 19th, 2016.
- the strain EG31 has been subjected to a batch directed evolution.
- this first medium named "GO xylose arabinose” and defined below, it is not the source of nitrogen is limiting but are the carbon sources.
- this medium contains 10 g / l of (NH 4) PO 4 but only 4 g / l of xylose and 5 g / l of arabinose.
- Medium 1 GO xylose arabinose (pH 5):
- the culture is carried out at 32 ° C. under a stirring of 100 rpm in flasks closed with stoppers which make it possible to reduce the supply of oxygen in the medium and to allow the C0 2 to escape under the overpressure which is produced all along. this culture.
- the cultivation lasts approximately 7 days under these conditions.
- the acid YF-glucose medium corresponds to the medium YF described above, containing 150 g / l of glucose and having a concentration of acetic acid (quantity introduced into the culture medium at pH 4.4) equal to 5 g / l.
- Medium 3 YF-xylose acid:
- the acid YF-xylose medium corresponds to the YF-xylose medium described above, with a concentration of acetic acid (quantity introduced into the culture medium at pH 5) equal to 4 g / l.
- the culture is carried out at 32 ⁇ 2 with a stirring of 100 rpm in flasks closed with stoppers which reduce the supply of oxygen in the medium and let out the C0 2 under pressure that is produced throughout this culture. .
- the cultivation lasts approximately 7 days under these conditions.
- the culture is carried out at 30 ° C. with stirring at 150 rpm in baffle flasks sealed with porous plugs which allow the supply of oxygen into the medium.
- the cultivation lasts approximately 24 to 48 hours under these conditions.
- Figure 9A shows the evolution of ethanol production as a function of the fermentation time for 6 of the best clones obtained.
- FIG. 9B shows the concentrations of arabinose in the fermentation medium after 160 hours of fermentation.
- the results confirm that all the clones selected at the end of the directed evolution consumed more arabinose than the EG31 strain, the clone noted C7 appearing as the most efficient.
- This clone was renamed EG32 strain which was deposited with the Institut Pasteur (National Collection of Cultures of Microorganisms, 25 rue du Dondel Roux, 75724 Paris Cedex 15) under number CNCM 1-5086 dated May 19, 2016.
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HRP20220572TT HRP20220572T1 (hr) | 2017-01-24 | 2018-01-24 | Dobivanje radnih sojeva kvasca za metaboliziranje arabinoze |
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FR1750550A FR3062134B1 (fr) | 2017-01-24 | 2017-01-24 | Obtention de souches de levure performantes pour la metabolisation de l'arabinose |
PCT/EP2018/051708 WO2018138135A1 (fr) | 2017-01-24 | 2018-01-24 | Obtention de souches de levure performantes pour la metabolisation de l'arabinose |
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CN113215203B (zh) * | 2021-02-26 | 2023-06-09 | 中粮生化能源(肇东)有限公司 | 共发酵酵母菌扩培发酵产乙醇的方法 |
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SE0202090D0 (sv) | 2002-05-08 | 2002-07-04 | Forskarpatent I Syd Ab | A modifierd yeast consuming L-arabinose |
CA2664646C (fr) * | 2006-10-02 | 2016-09-20 | Dsm Ip Assets B.V. | Genie metabolique de cellules de levure induisant la fermentation de l'arabinose |
DE102007016534B4 (de) | 2007-04-05 | 2019-03-28 | Butalco Gmbh | Neuer Expressionsvektor von Codon-optimierten Genen eines Arabinose-Stoffwechselweges zur Arabinose-Umsetzung in Hefe |
JP5219074B2 (ja) * | 2008-01-24 | 2013-06-26 | 独立行政法人産業技術総合研究所 | キシロース発酵能が優れた六炭糖・五炭糖同時発酵酵母およびそれを用いたエタノールの高効率生産方法 |
US20110104736A1 (en) * | 2008-03-13 | 2011-05-05 | Jacobus Thomas Pronk | Selection of organisms capable of fermenting mixed substrates |
DE102008031350B4 (de) | 2008-07-02 | 2011-02-10 | Johann Wolfgang Goethe-Universität Frankfurt am Main | Prokaryotische Xylose-Isomerase zur Konstruktion Xylose-vergärender Hefen |
UA108853C2 (uk) * | 2009-07-10 | 2015-06-25 | Спосіб ферментації галактози | |
FR2953857B1 (fr) | 2009-12-15 | 2012-10-12 | Lesaffre & Cie | Nouvelles souches de levure pour la production d'alcool |
CN102869766B (zh) * | 2010-04-21 | 2015-11-25 | 帝斯曼知识产权资产管理有限公司 | 适于发酵混合的糖组合物的细胞 |
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FR2968313B1 (fr) | 2010-12-03 | 2014-10-10 | Lesaffre & Cie | Procede de preparation d'une levure industrielle, levure industrielle et application a la production d'ethanol a partir d'au moins un pentose |
MX2013012269A (es) * | 2011-04-22 | 2013-11-22 | Dsm Ip Assets Bv | Celula de levadura capaz de convertir azucares que incluyen arabinosa y xilosa. |
AR087423A1 (es) * | 2011-08-04 | 2014-03-19 | Dsm Ip Assets Bv | Celula capaz de fermentar azucares pentosas |
FR2991340B1 (fr) | 2012-06-01 | 2016-02-19 | Lesaffre & Cie | Procede d'obtention de souches de levure ameliorees par modification genetique et croisement |
FR2991339B1 (fr) * | 2012-06-01 | 2016-02-19 | Lesaffre & Cie | Souches de levure aptes a metaboliser le xylose et resistantes a au moins un inhibiteur de fermentation, procede d'obtention et utilisation |
JP6236634B2 (ja) * | 2012-08-24 | 2017-11-29 | 国立大学法人神戸大学 | バイオマスからのエタノールの生産方法 |
WO2014207087A1 (fr) * | 2013-06-26 | 2014-12-31 | Abengoa Bioenergia Nuevas Tecnologias S.A. | Production de carburants avancés et de produits chimiques par des levures selon le concept de biocarburants de deuxième génération |
FR3017623B1 (fr) | 2014-02-17 | 2018-08-10 | Lesaffre Et Compagnie | Souche fermentant les pentoses a propagation optimisee |
FR3035405B1 (fr) * | 2015-04-27 | 2019-04-19 | Lesaffre Et Compagnie | Souche de levure presentant une capacite amelioree a fermenter le xylose en presence d'acide acetique |
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AU2018213056A1 (en) | 2019-07-25 |
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PT3574120T (pt) | 2022-05-13 |
CA3049415A1 (fr) | 2018-08-02 |
DK3574120T3 (da) | 2022-05-23 |
BR112019014115A2 (pt) | 2020-02-11 |
FR3062134B1 (fr) | 2023-07-21 |
ZA201904267B (en) | 2022-03-30 |
FR3062134A1 (fr) | 2018-07-27 |
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